N5-(L-1-carboxyethyl)-L-ornithine synthase (CEOS, EC 1.5.1.24) from Lactococcus lactis consists of four identical subunits of 35,300 MW. The enzyme catalyses an NADPH-dependent reductive condensation between pyruvate and the side-chain amino-group of L-ornithine or L-lysine, and may be important for post-translational modification of protein lysyl residues. Enzyme assays, intrinsic tyrosyl and tryptophanyl fluorescence, thiol group titration, hydrophobic group exposure, light scattering, far-UV circular dichroism, second-derivative UV absorption, size-exclusion chromatography and analytical ultracentrifugation were employed to monitor the denaturation and unfolding pathway of CEOS. At 0.2 M GdnHCl or 0.7 M NaCl, ca 1.6-fold enzyme activation was observed. At ca 1 M GdnHCl, CEOS was inactivated. A time-, temperature-, and concentration-dependent formation of soluble aggregates occurred at 0.5 M - 1.5 M GdnHCl concentrations due to noncovalent interactions arising from an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomers was observed between 2 and 3.5 M GdnHCl (without observable trimer or dimer intermediates), as evidenced by Tyr, Typ, and sulfhydryl group exposure, loss of secondary structure, gel filtration elution profiles, and sedimentation equilibrium. Dissociation and unfolding of tetrameric CEOS was concerted. Yields of reactivated CEOS by 100-fold dilution from 5 M GdnHCl were improved when dissociation and reconstitution took place at 0 rather than at 25 deg C, and were further improved as the concentration of CEOS was decreased. Refolding of unfolded subunits (30 micromolar) and tetramer assembly upon 100-fold dilution from 5 M GdnHCl at 0 deg C was increased ca 4-fold (to 28% reactivation) when incubated at 15 deg C for 3 h with the E. coli molecular chaperonin GroEL, Mg-ATP, 100 mM KCl, and 20 mM Tris, pH 7.2.